In spite of our knowledge of the Huntington's disease gene and of the disease mutations, and in spite of an enormous amount of creative work, the twin goals of a molecular level understanding of the disease mechanism, and the molecular therapy one hopes would emerge from this understanding, are not in reach. One difficulty holding the field back has been the unusual nature and unknown function of the disease gene product, a seemingly poorly behaved protein of over 3500 amino acids and no proven function. The full length Huntingtin protein has been very challenging to make and to work with, while the more manageable N- terminal fragment, exon1, though accessible by recombinant synthesis, is also poorly behaved in solution, especially when it contains a polyglutamine sequence in the pathological repeat length range of 37 or more. During previous terms of this grant, our group has taken a reductionist approach to this problem, working with peptide fragments that are accessible by chemical synthesis. Over the last funding period we moved from studies on simple polyglutamine (polyQ) sequences to studies of how the N and C-terminal flanking sequences of the polyQ in exon1 influence the solution conformation and aggregation of polyQ. These studies revealed a powerful and mechanistically complex accelerating effect by the 17 amino acid sequence on the N-terminal side of polyQ in exon1 (httNT) on polyQ aggregation along with a complete change in aggregation mechanism and the structures of the aggregated products. As part of our work to understand this effect, we developed the ability to produce chemically accessible analogs of htt N-terminal fragments (NTFs) related to exon1 that appear to behave similarly to authentic recombinant exon1, but at the same time are more manageable and amenable to introduction of small, well-defined probes for such techniques as NMR and FRET. In other recent work, we developed a staining method specific for amyloid forms of htt NTFs and polyQ in tissue and cells that has allowed us to visualize different aggregate structures in the cellular context. We also discovered, and engineered for cellular delivery, peptide-based inhibitors of different aggregation pathways of htt NTFs. In this renewal application, we propose to exploit these tools to reach a new understanding of the molecular events that are triggered by expanded polyQ in htt. The current wisdom in the HD field is that htt must be proteolytically processed to an N-terminal fragment as an early step in the disease mechanism. The physical state that NTF takes when it becomes toxic remains a mystery. This toxic physical state may be a misfolded form of the monomeric fragment, a short-lived aggregation intermediate, or a stable aggregate such as an amyloid fibril. In this proposal we will focus on structural analysis of monomers and stable aggregates, using chemically synthesized and recombinant htt NTFs including exon1, with biophysical tools such as NMR, fluorescence resonance energy transfer, hydrogen-deuterium exchange, and mutational analysis. We will also conduct a detailed study of the polyQ repeat length effect on the structure of the monomeric htt NTF as well as its aggregation propensity, and compare both to the repeat length dependence of HD risk. We will also explore the toxicities of these physical states by testing in vitro-produced materials and by probing the effect of inhibitors on htt-producing cells.